Modulators of hepatocyte growth factor activator

ABSTRACT

The invention provides methods and compositions for modulating hepatocyte growth factor activator function.

RELATED APPLICATIONS

This application is a non-provisional application filed under 37 CFR1.53(b)(1), claiming priority under 35 USC 119(e) to provisionalapplication No. 60/615,657 filed Oct. 4, 2004, the contents of which areincorporated herein in their entirety by reference.

TECHNICAL FIELD

The present invention relates generally to the fields of molecularbiology and growth factor regulation. More specifically, the inventionconcerns modulators of hepatocyte growth factor activator function, anduses of said modulators.

BACKGROUND

Hepatocyte growth factor (HGF) promotes cell proliferation, migration,angiogenesis, survival and morphogenesis by activating the receptortyrosine kinase Met (reviewed in 8, 9). In addition to its importance innormal physiology, the HGF/Met pathway has been implicated in invasivetumor growth and tumor metastasis (8). HGF has high similarity to theserine protease plasminogen and is composed of a α-chain containing anN-domain and four Kringle domains and a β-chain with homology tochymotrypsin-like proteases. It is secreted into the extracellularmatrix as an inactive single chain precursor (pro-HGF) and requiresactivation cleavage at Arg494-Val495 to form the biologically competent,disulfide-linked α/β heterodimer (10-13). This step is mediated bypro-HGF converting serine proteases, such as hepatocyte growth factoractivator (HGFA) (14). HGFA is inhibited by cell surface-expressedKunitz-type inhibitors, such as the two hepatocyte growth factoractivator inhibitor splice variants HAI-1 (16-17) and HAI-1B (15) and byHAI-2 (18). HAI-2 (also known as placental bikunin) (19) also potentlyinhibits factor XIa and plasma kallikrein (20), whereas HAI-1B haslittle or no inhibitory activity (15). Therefore, the biologicalavailability of the pro-HGF pool in the extracellular matrix isregulated by the activities of pro-HGF convertases such as HGFA andtheir inhibitors.

Since activation of pro-HGF requires cleavage by a convertase such asHGFA, modulation of HGFA function and/or its interaction with itssubstrate could prove to be an efficacious therapeutic approach. In thisregard, there is a clear need to identify clinically relevant agentscapable of modulating activity of and/or specificifically interactingwith HGFA. The invention fulfills this need and provides other benefits.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

DISCLOSURE OF THE INVENTION

The invention provides methods, compositions, kits and articles ofmanufacture for modulating hepatocyte growth factor activator (HGFA)function, thereby modulating physiological effects of HGFA activity.Modulation of HGFA function can be effected by the use of antibodies asdescribed herein.

The invention provides modulator molecules capable of use for modulatingHGFA function. In one embodiment, HGFA function is modulated throughinhibition of HGFA activity (e.g., proteolytic activity). Generally, themodulator molecules comprise an antibody as described herein. Themodulator molecules are capable of effecting modulation either directly(e.g., by binding to HGFA and interefering with HGFA proteolyticactivity) or indirectly (e.g., by targeting/directing an active agent toHGFA in a tissue or cell, wherein the active agent is capable ofinterfering with HGFA proteolytic activity). In one embodiment, theinvention provides an antagonist molecule comprising an antibody thatbinds to HGFA. In one embodiment, binding of the antagonist to HGFAinterferes with HGFA proteolytic activity. In one embodiment, binding ofthe antagonist to HGFA interferes with activation of HGF by HGFA. In oneembodiment, the antibody binds to the active site of HGFA. In oneembodiment, the antibody binds to HGFA at a position other than the HGFAactive site (e.g., an exosite). In one embodiment, binding of theantibody to HGFA at a position other than the HGFA active site inhibitsinteraction of HGFA with its substrate molecule. In one embodiment,binding of the antibody to HGFA at a position other than the HGFA activesite inhibits HGFA proteolytic activity.

In one aspect, the invention provides antagonists that disrupt theHGF/c-met signaling pathway. For example, the invention provides amolecule that inhibits HGFA cleavage of proHGF (e.g., cleavage at theR494-V495 position). The molecule can exert its inhibitory function inany number of ways, including but not limited to binding to HGFA at itsactive site and/or at a site other than the active site (e.g., anexosite) such that HGFA cleavage of proHGF is inhibited. The moleculecan bind to HGFA in complexed or uncomplexed form. The molecule can alsoexert its inhibitory function by interfering with one or more aspects ofthe HGF activation process. For example, in one embodiment, anantagonist molecule of the invention binds to HGFA-proHGF complex suchthat cleavage of proHGF is inhibited. In one embodiment, binding of themolecule to proHGF or HGFA (singly or in complex) inhibits release ofHGF subsequent to cleavage by HGFA. In one embodiment, an antagonistmolecule of the invention does not inhibit HGF binding to c-met. Forexample, in one embodiment, an antagonist molecule of the invention isnot an antibody or fragment thereof having similar inhibitory and/orbinding ability as the antibody produced by hybridoma cell linedeposited under American Type Culture Collection Accession Number ATCCHB-11894 (hybridoma 1A3.3.13) or HB-11895 (hybridoma 5D5.11.6). In oneembodiment, an antagonist molecule of the invention inhibits biologicalactivities associated with HGF/c-met activation.

In one aspect, the invention provides an antibody comprising a CDR-H1region comprising the sequence of SEQ ID NO:3, 6, 9, 12, 15, 18, 21, 24,27, 30, 33, 36, 39 or 42. In one aspect, the invention provides anantibody comprising a CDR-H2 region comprising the sequence of SEQ IDNO:4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40 or 43. In oneaspect, the invention provides an antibody comprising a CDR-H3 regioncomprising the sequence of SEQ ID NO:5, 8, 11, 14, 17, 20, 23, 26, 29,32, 35, 38, 41 or 44. In one embodiment, the invention provides anantibody comprising a CDR-H1 region comprising the sequence of SEQ IDNO:3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39 or 42, and a CDR-H2region comprising the sequence of SEQ ID NO:4, 7, 10, 13, 16, 19, 22,25, 28, 31, 34, 37, 40 or 43. In one embodiment, the invention providesan antibody comprising a CDR-H1 region comprising the sequence of SEQ IDNO:3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39 or 42, and a CDR-H3region comprising the sequence of SEQ ID NO:5, 8, 11, 14, 17, 20, 23,26, 29, 32, 35, 38, 41 or 44. In one embodiment, the invention providesan antibody comprising a CDR-H2 region comprising the sequence of SEQ IDNO:4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40 or 43, and a CDR-H3region comprising the sequence of SEQ ID NO:5, 8, 11, 14, 17, 20, 23,26, 29, 32, 35, 38, 41 or 44. In one embodiment, the invention providesan antibody comprising a CDR-H1 region comprising the sequence of SEQ IDNO:3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39 or 42, a CDR-H2region comprising the sequence of SEQ ID NO:4, 7, 10, 13, 16, 19, 22,25, 28, 31, 34, 37, 40 or 43, and a CDR-H3 region comprising thesequence of SEQ ID NO:5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41or 44.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three of the following:

(i) a CDR-H1 sequence comprising the sequence of SEQ ID NO:3;

(ii) a CDR-H2 sequence comprising the sequence of SEQ ID NO:4;

(iii) a CDR-H3 sequence comprising the sequence of SEQ ID NO:5.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three of the following:

(i) a CDR-H1 sequence comprising the sequence of SEQ ID NO:6;

(ii) a CDR-H2 sequence comprising the sequence of SEQ ID NO:7;

(iii) a CDR-H3 sequence comprising the sequence of SEQ ID NO:8.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three of the following:

(i) a CDR-H1 sequence comprising the sequence of SEQ ID NO:9;

(ii) a CDR-H2 sequence comprising the sequence of SEQ ID NO:10;

(iii) a CDR-H3 sequence comprising the sequence of SEQ ID NO:11.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three of the following:

(i) a CDR-H1 sequence comprising the sequence of SEQ ID NO:12;

(ii) a CDR-H2 sequence comprising the sequence of SEQ ID NO:13;

(iii) a CDR-H3 sequence comprising the sequence of SEQ ID NO: 14.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three of the following:

(i) a CDR-H1 sequence comprising the sequence of SEQ ID NO:15;

(ii) a CDR-H2 sequence comprising the sequence of SEQ ID NO:16;

(iii) a CDR-H3 sequence comprising the sequence of SEQ ID NO:17.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three of the following:

(i) a CDR-H1 sequence comprising the sequence of SEQ ID NO:18;

(ii) a CDR-H2 sequence comprising the sequence of SEQ ID NO:19;

(iii) a CDR-H3 sequence comprising the sequence of SEQ ID NO:20.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three of the following:

(i) a CDR-H1 sequence comprising the sequence of SEQ ID NO:21;

(ii) a CDR-H2 sequence comprising the sequence of SEQ ID NO:22;

(iii) a CDR-H3 sequence comprising the sequence of SEQ ID NO:23.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three of the following:

(i) a CDR-H1 sequence comprising the sequence of SEQ ID NO:24;

(ii) a CDR-H2 sequence comprising the sequence of SEQ ID NO:25;

(iii) a CDR-H3 sequence comprising the sequence of SEQ ID NO:26.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three of the following:

(i) a CDR-H1 sequence comprising the sequence of SEQ ID NO:27;

(ii) a CDR-H2 sequence comprising the sequence of SEQ ID NO:28;

(iii) a CDR-H3 sequence comprising the sequence of SEQ ID NO:29.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three of the following:

(i) a CDR-H1 sequence comprising the sequence of SEQ ID NO:30;

(ii) a CDR-H2 sequence comprising the sequence of SEQ ID NO:31;

(iii) a CDR-H3 sequence comprising the sequence of SEQ ID NO:32.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three of the following:

(i) a CDR-H1 sequence comprising the sequence of SEQ ID NO:33;

(ii) a CDR-H2 sequence comprising the sequence of SEQ ID NO:34;

(iii) a CDR-H3 sequence comprising the sequence of SEQ ID NO:35.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three of the following:

(i) a CDR-H1 sequence comprising the sequence of SEQ ID NO:36;

(ii) a CDR-H2 sequence comprising the sequence of SEQ ID NO:37;

(iii) a CDR-H3 sequence comprising the sequence of SEQ ID NO:38.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three of the following:

(i) a CDR-H1 sequence comprising the sequence of SEQ ID NO:39;

(ii) a CDR-H2 sequence comprising the sequence of SEQ ID NO:40;

(iii) a CDR-H3 sequence comprising the sequence of SEQ ID NO:41.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three of the following:

(i) a CDR-H1 sequence comprising the sequence of SEQ ID NO:42;

(ii) a CDR-H2 sequence comprising the sequence of SEQ ID NO:43;

(iii) a CDR-H3 sequence comprising the sequence of SEQ ID NO:44.

The amino acid sequences of SEQ ID NOs:3-44 are numbered with respect toindividual CDR (i.e., H1, H2 or H3) as indicated in FIG. 1, thenumbering being consistent with the Kabat numbering system as describedbelow.

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain CDR sequence(s) comprising the sequence of at least one,at least two, or all three of the H1 (SEQ ID NO: 71-84), H2 (SEQ ID NO:85-98) and/or H3 (SEQ ID NO: 99-112) sequences for each clone depictedin FIGS. 1B, 1C and 1D.

In one aspect, the invention provides antibodies comprising heavy chainCDR sequences as depicted in FIGS. 1A, B, C and D. In some embodiment,these antibodies further comprise a light chain variable domain ofhumanized 4D5 antibody (huMAb4D5-8) (HERCEPTIN®, Genentech, Inc., SouthSan Francisco, Calif., USA) (also referred to in U.S. Pat. No. 6,407,213and Lee et al., J. Mol. Biol. (2004), 340(5):1073-93) as depicted in SEQID NO:45 below. (SEQ ID NO:45) Asp Ile Gln Met Thr Gln Ser Pro Ser SerLeu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln AspVal Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys LeuLeu Ile Tyr Ser Ala Ser Phe Leu Glu Ser Gly Val Pro Ser Arg Phe Ser GlySer Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu AspPhe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly GlnGly Thr Lys Val Glu Ile Lys Arg Thr

In one embodiment, the huMAb4D5-8 light chain variable domain sequenceis modified at one or more of positions 30, 66 and 91 (Asn, Arg and Hisas indicated in bold/italics above, respectively). In one embodiment,the modified huMAb4D5-8 sequence comprises Ser in position 30, Gly inposition 66 and/or Ser in position 91. Accordingly, in one embodiment,an antibody of the invention comprises a light chain variable domaincomprising the sequence depicted in SEQ ID NO: 54 below: (SEQ ID NO: 54)1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly AspArg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val

 Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu IleTyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe AlaThr Tyr Tyr Cys Gln Gln

 Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 107(CDR residues are underlined)

Substituted residues with respect to huMAb4D5-8 are indicated inbold/italics above.

Antibodies of the invention can further comprise any suitable frameworkand/or light chain variable domain sequences, provided HGFA bindingactivity is substantially retained. For example, in some embodiments,these antibodies further comprise a human subgroup III heavy chainframework consensus sequence. In one embodiment of these antibodies, theframework consensus sequence comprises substitution at position 71, 73and/or 78. In some embodiments of these antibodies, position 71 is A, 73is T and/or 78 is A. In one embodiment, these antibodies comprise heavychain variable domain framework sequences of humanized 4D5 antibody(huMAb 4D5-8) (HERCEPTIN®, Genentech, Inc., South San Francisco, Calif.,USA) (also referred to in U.S. Pat. No. 6,407,213 and Lee et al., J.Mol. Biol. (2004), 340(5):1073-93). In one embodiment, the humanized4D5-8 antibody is as described in U.S. Pat. No. 6,407,213. In oneembodiment, these antibodies further comprise a human κI light chainframework consensus sequence. In one embodiment, these antibodiescomprise light chain variable domain sequences of humanized 4D5 antibody(huMAb 4D5-8) (SEQ ID NO:45) (HERCEPTIN®, Genentech, Inc., South SanFrancisco, Calif., USA) (also referred to in U.S. Pat. No. 6,407,213 andLee et al., J. Mol. Biol. (2004), 340(5):1073-93), or the modifiedvariant thereof as depicted in SEQ ID NO: 54.

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain, wherein the framework sequence comprises the sequencesof SEQ ID NO: 46, 47, 48 and 49 (FR1, 2, 3, and 4, respectively), andCDR H1, H2 and H3 sequences as depicted in FIGS. 1A, B, C, and/or D. Inone embodiment, an antibody of the invention comprises a light chainvariable domain, wherein the framework sequence comprises the sequenceof SEQ ID NO: 50, 51, 52 and 53 (FR1, 2, 3, and 4, respectively), andCDR L1, L2 and L3 sequences as depicted in SEQ ID NO: 54.

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain, wherein the framework sequence comprises the sequenceof SEQ ID NO: 59, 60, 61 and 62 (FR1, 2, 3 and 4, respectively) (FIG.1E), and CDR H1, H2 and H3 sequences as depicted in FIG. 1. In oneembodiment, an antibody of the invention comprises a light chainvariable domain, wherein the framework sequence comprises the sequenceof SEQ ID NO: 55, 56, 57, and 58 (FR 1, 2, 3 and 4, respectively) (FIG.1E), and CDR L1, L2 and L3 sequences as depicted in SEQ ID NO: 54.

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain, wherein the framework sequence comprises the sequenceof SEQ ID NO: 67, 68, 69 and 70 (FR 1, 2, 3 and 4, respectively) (FIG.1F), and CDR H1, H2 and H3 sequences as depicted in FIGS. 1A, B, Cand/or D. In one embodiment, an antibody of the invention comprises alight chain variable domain, wherein the framework sequence comprisesthe sequence of SEQ ID NO: 63, 64, 65, and 66 (FR 1, 2, 3 and 4,respectively) (FIG. 1F), and CDR L1, L2 and L3 sequences as depicted inSEQ ID NO: 54.

In one aspect, the invention provides an antibody that competes with anyof the above-mentioned antibodies for binding to HGFA. In one aspect,the invention provides an antibody that binds to the same epitope onHGFA as any of the above-mentioned antibodies. In one embodiment, anantibody of the invention is affinity matured, humanized, chimeric, orhuman. In one embodiment, an antibody of the invention is an antibodyfragment (as described herein), or a substantially full length antibody.In one embodiment, an antibody of the invention comprises a wild type Fcregion, or a variant thereof. In one embodiment, an antibody of theinvention is an IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgE or IgD.

In one aspect, an antagonist molecule of the invention is linked to atoxin such as a cytotoxic agent. These molecules/substances can beformulated or administered in combination with an additive/enhancingagent, such as a radiation and/or chemotherapeutic agent.

The invention also provides methods and compositions useful formodulating disease states associated with dysregulation of the HGF/c-metsignaling axis. Thus, in one aspect, the invention provides a method ofmodulating c-met activation in a subject, said method comprisingadministering to the subject a modulator molecule of the invention thatinhibits HGFA cleavage of proHGF, whereby c-met activation is modulated.In one aspect, the invention provides a method of treating apathological condition associated with activation of c-met in a subject,said method comprising administering to the subject a modulator moleculeof the invention that inhibits HGFA cleavage of proHGF, whereby c-metactivation is inhibited. In one embodiment, the modulator molecule ofthe invention is an antibody that binds to HGFA.

The HGF/c-met signaling pathway is involved in multiple biological andphysiological functions, including, e.g., cell growth stimulation (e.g.cell proliferation, cell survival, cell migration, cell morphogenesis)and angiogenesis. Thus, in another aspect, the invention provides amethod of inhibiting c-met activated cell growth (e.g. proliferationand/or survival), said method comprising contacting a cell or tissuewith an antagonist of the invention, whereby cell proliferationassociated with c-met activation is inhibited. In yet another aspect,the invention provides a method of inhibiting angiogenesis, said methodcomprising administering to a cell, tissue, and/or subject with acondition associated with abnormal angiogenesis an antagonist of theinvention, whereby angiogenesis is inhibited.

In one aspect, the invention provides use of a modulator molecule of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as a cancer, a tumor, a cellproliferative disorder, an immune (such as autoimmune) disorder and/oran angiogenesis-related disorder.

In one aspect, the invention provides use of a nucleic acid of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as a cancer, a tumor, a cellproliferative disorder, an immune (such as autoimmune) disorder and/oran angiogenesis-related disorder.

In one aspect, the invention provides use of an expression vector of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as a cancer, a tumor, a cellproliferative disorder, an immune (such as autoimmune) disorder and/oran angiogenesis-related disorder.

In one aspect, the invention provides use of a host cell of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as a cancer, a tumor, a cellproliferative disorder, an immune (such as autoimmune) disorder and/oran angiogenesis-related disorder.

In one aspect, the invention provides use of an article of manufactureof the invention in the preparation of a medicament for the therapeuticand/or prophylactic treatment of a disease, such as a cancer, a tumor, acell proliferative disorder, an immune (such as autoimmune) disorderand/or an angiogenesis-related disorder.

In one aspect, the invention provides use of a kit of the invention inthe preparation of a medicament for the therapeutic and/or prophylactictreatment of a disease, such as a cancer, a tumor, a cell proliferativedisorder, an immune (such as autoimmune) disorder and/or anangiogenesis-related disorder

In one aspect, the invention provides a method of inhibiting c-metactivated cell proliferation, said method comprising contacting a cellor tissue with an effective amount of a modulator molecule of theinvention, whereby cell proliferation associated with c-met activationis inhibited.

In one aspect, the invention provides a method of treating apathological condition associated with dysregulation of c-met activationin a subject; said method comprising administering to the subject aneffective amount of a modulator molecule of the invention, whereby saidcondition is treated.

In one aspect, the invention provides a method of inhibiting the growthof a cell that expresses c-met or hepatocyte growth factor, or both,said method comprising contacting said cell with a modulator molecule ofthe invention thereby causing an inhibition of growth of said cell. Inone embodiment, the cell is contacted by HGF expressed by a differentcell (e.g., through a paracrine effect).

In one aspect, the invention provides a method of therapeuticallytreating a mammal having a cancerous tumor comprising a cell thatexpresses c-met or hepatocyte growth factor, or both, said methodcomprising administering to said mammal an effective amount of an amodulator molecule of the invention, thereby effectively treating saidmammal. In one embodiment, the cell is contacted by HGF expressed by adifferent cell (e.g., through a paracrine effect).

In one aspect, the invention provides a method for treating orpreventing a cell proliferative disorder associated with increasedexpression or activity of HGFA, said method comprising administering toa subject in need of such treatment an effective amount of an amodulator molecule of the invention, thereby effectively treating orpreventing said cell proliferative disorder. In one embodiment, saidproliferative disorder is cancer.

In one aspect, the invention provides a method for treating orpreventing a cell proliferative disorder associated with increasedexpression or activity of c-met or hepatocyte growth factor, or both,said method comprising administering to a subject in need of suchtreatment an effective amount of a modulator molecule of the invention,thereby effectively treating or preventing said cell proliferativedisorder. In one embodiment, said proliferative disorder is cancer.

In one aspect, the invention provides a method for inhibiting the growthof a cell, wherein growth of said cell is at least in part dependentupon a growth potentiating effect of HGFA, said method comprisingcontacting said cell with an effective amount of a modulator molecule ofthe invention, thereby inhibiting the growth of said cell. In oneembodiment, the cell is contacted by HGF expressed by a different cell(e.g., through a paracrine effect).

In one aspect, the invention provides a method for inhibiting the growthof a cell, wherein growth of said cell is at least in part dependentupon a growth potentiating effect of c-met or hepatocyte growth factor,or both, said method comprising contacting said cell with an effectiveamount of a modulator molecule of the invention, thereby inhibiting thegrowth of said cell. In one embodiment, the cell is contacted by HGFexpressed by a different cell (e.g., through a paracrine effect).

In one aspect, the invention provides a method of therapeuticallytreating a tumor in a mammal, wherein the growth of said tumor is atleast in part dependent upon a growth potentiating effect of HGFA, saidmethod comprising contacting said cell with an effective amount of amodulator molecule of the invention, thereby effectively treating saidtumor. In one embodiment, the cell is contacted by HGF expressed by adifferent cell (e.g., through a paracrine effect).

In one aspect, the invention provides a method of therapeuticallytreating a tumor in a mammal, wherein the growth of said tumor is atleast in part dependent upon a growth potentiating effect of c-met orhepatocyte growth factor, or both, said method comprising contactingsaid cell with an effective amount of a modulator molecule of theinvention, thereby effectively treating said tumor. In one embodiment,the cell is contacted by HGF expressed by a different cell (e.g.,through a paracrine effect).

Methods of the invention can be used to affect any suitable pathologicalstate, for example, cells and/or tissues associated with dysregulationof the HGF/c-met signaling pathway, e.g. through increased HGF activityassociated with HGFA activation of HGF. In one embodiment, a cell thatis targeted in a method of the invention is a cancer cell. For example,a cancer cell can be one selected from the group consisting of a breastcancer cell, a colorectal cancer cell, a lung cancer cell, a papillarycarcinoma cell (e.g., of the thyroid gland), a colon cancer cell, apancreatic cancer cell, an ovarian cancer cell, a cervical cancer cell,a central nervous system cancer cell, an osteogenic sarcoma cell, arenal carcinoma cell, a hepatocellular carcinoma cell, a bladder cancercell, a prostate cancer cell, a gastric carcinoma cell, a head and necksquamous carcinoma cell, a melanoma cell and a leukemia cell. In oneembodiment, a cell that is targeted in a method of the invention is ahyperproliferative and/or hyperplastic cell. In one embodiment, a cellthat is targeted in a method of the invention is a dysplastic cell. Inyet another embodiment, a cell that is targeted in a method of theinvention is a metastatic cell.

Methods of the invention can further comprise additional treatmentsteps. For example, in one embodiment, a method further comprises a stepwherein a targeted cell and/or tissue (e.g., a cancer cell) is exposedto radiation treatment or a chemotherapeutic agent.

As described herein, HGF/c-met activation is an important biologicalprocess the dysregulation of which leads to numerous pathologicalconditions. Accordingly, in one embodiment of methods of the invention,a cell that is targeted (e.g., a cancer cell) is one in which activationof HGF/c-met is enhanced as compared to a normal cell of the same tissueorigin. In one embodiment, a method of the invention causes the death ofa targeted cell. For example, contact with a modulator molecule of theinvention may result in a cell's inability to signal through the c-metpathway, which results in cell death.

Dysregulation of c-met activation (and thus signaling) can result from anumber of cellular changes, including, for example, overexpression ofHGF (c-met's cognate ligand) and/or HGFA, and/or increased activation ofHGF by HGFA. Accordingly, in some embodiments, a method of the inventioncomprises targeting a tissue wherein one or more of HGFA, c-met andhepatoctye growth factor, is more abundantly expressed and/or present(e.g., a cancer) as compared to a normal tissue of the same origin. AnHGF or c-met-expressing cell can be regulated by HGFA from a variety ofsources, i.e. in an autocrine or paracrine manner. For example, in oneembodiment of methods of the invention, a targeted cell iscontacted/bound by hepatocyte growth factor activated by HGFA expressedin a different cell (e.g., via a paracrine effect). Said different cellcan be of the same or of a different tissue origin. In one embodiment, atargeted cell is contacted/bound by HGF activated by HGFA expressed bythe targeted cell itself (e.g., via an autocrine effect/loop).

In one aspect, the invention provides compositions comprising one ormore modulator molecules of the invention and a carrier. In oneembodiment, the carrier is pharmaceutically acceptable.

In one aspect, the invention provides nucleic acids encoding a modulatormolecule of the invention. In one embodiment, a nucleic acid of theinvention encodes a modulator molecule which is or comprises an antibodyor fragment thereof.

In one aspect, the invention provides vectors comprising a nucleic acidof the invention.

In one aspect, the invention provides host cells comprising a nucleicacid or a vector of the invention. A vector can be of any type, forexample a recombinant vector such as an expression vector. Any of avariety of host cells can be used. In one embodiment, a host cell is aprokaryotic cell, for example, E. coli. In one embodiment, a host cellis a eukaryotic cell, for example a mammalian cell such as ChineseHamster Ovary (CHO) cell.

In one aspect, the invention provides methods for making a modulatormolecule of the invention. For example, the invention provides a methodof making a modulator molecule which is or comprises an antibody (orfragment thereof), said method comprising expressing in a suitable hostcell a recombinant vector of the invention encoding said antibody (orfragment thereof), and recovering said antibody.

In one aspect, the invention provides an article of manufacturecomprising a container; and a composition contained within thecontainer, wherein the composition comprises one or more modulatormolecules of the invention. In one embodiment, the composition comprisesa nucleic acid of the invention. In one embodiment, a compositioncomprising a modulator molecule further comprises a carrier, which insome embodiments is pharmaceutically acceptable. In one embodiment, anarticle of manufacture of the invention further comprises instructionsfor administering the composition (for e.g., the modulator molecule) toa subject.

In one aspect, the invention provides a kit comprising a first containercomprising a composition comprising one or more modulator molecules ofthe invention; and a second container comprising a buffer. In oneembodiment, the buffer is pharmaceutically acceptable. In oneembodiment, a composition comprising a modulator molecule furthercomprises a carrier, which in some embodiments is pharmaceuticallyacceptable. In one embodiment, a kit further comprises instructions foradministering the composition (for e.g., the modulator molecule) to asubject.

In one aspect the invention provides a method of diagnosing a diseasecomprising determining the level of HGFA in a test sample of tissuecells by contacting the sample with an antibody of the invention,whereby HGFA bound by the antibody indicates presence and/or amount ofHGFA in the sample. In another aspect, the invention provides a methodof determining whether an individual is at risk for a disease comprisingdetermining the level of HGFA in a test sample of tissue cell bycontacting the test sample with an antibody of the invention and therebydetermining the amount of HGFA present in the sample, wherein a higherlevel of HGFA in the test sample, as compared to a control samplecomprising normal tissue of the same cell origin as the test sample, isan indication that the individual is at risk for the disease. In oneembodiment of methods of the invention, the level of HGFA is determinedbased on amount of HGFA polypeptide indicated by amount of HGFA bound bythe antibody in the test sample. An antibody employed in the method mayoptionally be detectably labeled, attached to a solid support, or thelike.

In one aspect, the invention provides a method of binding an antibody ofthe invention to HGFA present in a bodily fluid, for example blood.

In yet another aspect, the invention is directed to a method of bindingan antibody of the invention to a cell that expresses and/or isresponsive to HGFA, wherein the method comprises contacting said cellwith said antibody under conditions which are suitable for binding ofthe antibody to HGFA and allowing binding therebetween. In oneembodiment, binding of said antibody to HGFA on the cell inhibits anHGFA biological function. In one embodiment, said antibody does notinhibit interaction of HGFA with its substrate molecule. In oneembodiment, said antibody binds to an HGFA molecule on the cell andinhibits binding of another molecule (such as pro-HGF) to the HGFAmolecule.

In one aspect, the invention provides a method of targeting atherapeutic agent to an HGFA-associated tissue in a host, the methodcomprising administering to the host said therapeutic agent in a formthat is linked to an antibody of the invention, whereby the agent istargeted to the HGFA-associated tissue in the host. In one embodiment,the antibody that binds HGFA is capable of specifically binding to HGFAlocated on a cell (either in vitro or in vivo), for example where HGFAis present on the surface of a cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (A) Heavy chain CDR loop sequences of anti-HGFA antibodies. Thefigure shows the heavy chain CDR sequences, H1, H2, and H3. The lightchain sequence is humanized 4D5 sequence (see Lee et al., supra).Sequence numbering is as follows: clone 33 (CDRH1 is SEQ ID NO:3; CDRH2is SEQ ID NO:4; CDRH3 is SEQ ID NO:5); clone 35 (CDRH1 is SEQ ID NO:6;CDRH2 is SEQ ID NO:7; CDRH3 is SEQ ID NO:8); clone 37 (CDRH1 is SEQ IDNO:9; CDRH2 is SEQ ID NO:10; CDRH3 is SEQ ID NO:11); clone 39 (CDRH1 isSEQ ID NO:12; CDRH2 is SEQ ID NO:13; CDRH3 is SEQ ID NO:14); clone 42(CDRH1 is SEQ ID NO:15; CDRH2 is SEQ ID NO:16; CDRH3 is SEQ ID NO:17);clone 49 (CDRH1 is SEQ ID NO:18; CDRH2 is SEQ ID NO:19; CDRH3 is SEQ IDNO:20); clone 58 (CDRH1 is SEQ ID NO:21; CDRH2 is SEQ ID NO:22; CDRH3 isSEQ ID NO:23); clone 61 (CDRH1 is SEQ ID NO:24; CDRH2 is SEQ ID NO:25;CDRH3 is SEQ ID NO:26); clone 74 (CDRH1 is SEQ ID NO:27; CDRH2 is SEQ IDNO:28; CDRH3 is SEQ ID NO:29); clone 75 (CDRH1 is SEQ ID NO:30; CDRH2 isSEQ ID NO:31; CDRH3 is SEQ ID NO:32); clone 86 (CDRH1 is SEQ ID NO:33;CDRH2 is SEQ ID NO:34; CDRH3 is SEQ ID NO:35); clone 90 (CDRH1 is SEQ IDNO:36; CDRH2 is SEQ ID NO:37; CDRH3 is SEQ ID NO:38); clone 91 (CDRH1 isSEQ ID NO:39; CDRH2 is SEQ ID NO:40; CDRH3 is SEQ ID NO:41); clone 95(CDRH1 is SEQ ID NO:42; CDRH2 is SEQ ID NO:43; CDRH3 is SEQ ID NO:44).Amino acid positions are numbered according to the Kabat numberingsystem as described below. IC50 values are also indicated in the last(right hand) column.

-   -   (B), (C) and (D) Heavy chain CDR loop sequences of anti-HGFA        antibodies.    -   (E) and (F) Exemplary framework region sequences. (E) HuMAb4D5-8        framework region sequences. (F) HuMAb4D5-8 framework region        sequences comprising modifications.

FIG. 2 Inhibition of HGFA-mediated proHGF activation by anti-HGFAantibodies. HGFA was incubated with ¹²⁵I-labelled proHGF and anti-HGFAantibodies for 4 hr at 37° C. Reactant concentrations were 50 μg/mlproHGF, 2 nM HGFA and 0.1 mg/ml (0.67 μM) antibodies. Aliquots wereanalyzed by SDS-PAGE under reducing conditions. Soluble HAI-1B (sHAI-1B)was used as a control inhibitor at 1 μM final concentration. A. Lane 1:(t=0) is aliquot taken at beginning of reaction, lane 2: no inhibitor,lane 3: sHAI-1B (1 μM), lane 4: #33, lane 5: #35, lane 6: #39, lane 7:#49, lane 8: #74, lane 9: #61. B. Lane 1: #42, lane 2: #91, lane 3: 58,lane 4: #37, lane 5: #75, lane 6: #90, lane 7: #86, lane 8: #95.

FIG. 3. Potent inhibition of HGFA-mediated proHGF conversion by antibody#58. Three different concentrations of the antibody #58 and thenon-blocking antibody #49 were used in ¹²⁵I-labelled proHGF conversionexperiments carried out as described in FIG. 1. Lane 1: (t=0) is aliquottaken at beginning of reaction, lane 2: no inhibitor, lane 3: sHAI-1B (1μM), lane 4: 0.67 μM Ab#49, lane 5: 0.13 μM Ab#49, lane 6: 0.03 μMAb#49, lane 7: 0.671 μM Ab#58, lane 8: 0.13 μM Ab#58, lane 9: 0.03 μMAb#58.

FIG. 4. Concentration-dependent inhibition of HGFA amidolytic activityby anti-HGFA antibodies 58 and 75. Various concentrations of antibodieswere incubated with HGFA (5 nM final concentration) in HBSA buffer for20 min at room temperature. After addition of Spectrozyme® fVIIa (200 μMfinal conc., K_(M)=200 μM) the linear rates of substrate activation weremeasured on a kinetic microplate reader. Inhibition of enzyme activitywas expressed as fractional activity (vi/vo) of uninhibited activity.

FIG. 5. Inhibition of HGFA amidolytic activity by IV49C and a smallmolecule active site binder/inhibitor. Various concentrations ofinhibitors were incubated with HGFA (2.5 nM for IV-49C and 5 nM for thesmall molecule, respectively) in HBSA buffer for 20 min at roomtemperature. Enzyme inhibition of Spectrozyme® fVIIa activation wasmeasured as described in FIG. 4. A. Inhibition by Kunitz domaininhibitor IV49C (filled circles) in comparison to the specific factorXIIa inhibitor corn trypsin inhibitor (open circles). B. Inhibition bythe small molecule inhibitor (filled triangles).

FIG. 6 Surface plasmon resonance measurements of HGFA binding toanti-HGFA antibodies #58 and #75. Anti-HGFA antibodies (full lengthIgG1) were immobilized on BIAcore chips and binding data were collectedfrom various concentrations of HGFA. For competition binding studies,HGFA (70 nM) was preincubated with various concentrations of sHAI-1B,IV-49C or small molecule active site binder. A-D: Binding of HGFA toantibody #58 (A) in the absence of inhibitor, or in the presence of (B)sHAI-1B, (C) IV-49C and (D) small molecule active site binder. E-H:Binding of HGFA to antibody #75 (E) in the absence of inhibitor, or inthe presence of (F) sHAI-1B, (G) IV49C and (H) small molecule activesite binder.

FIG. 7 Sequences of human (top line; SEQ ID NO:1) and murine (bottomline; SEQ ID NO:2) HGFA protein sequences.

FIG. 8 Table showing data related to inhibition of HGFA enzymaticactivity by various anti-HGFA antibodies.

FIG. 9 Table showing data related to binding of HGFA to anti-HGFAantibodies.

MODES FOR CARRYING OUT THE INVENTION

The invention provides methods, compositions, kits and articles ofmanufacture comprising modulators of hepatocyte growth factor activatorfunction, including methods of using such modulators.

Details of these methods, compositions, kits and articles of manufactureare provided herein.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel etal., eds., 1987, and periodic updates); “PCR: The Polymerase ChainReaction”, (Mullis et al., ed., 1994); “A Practical Guide to MolecularCloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual”(Barbas et al., 2001).

Definitions

The term “hepatocyte growth factor activator” or “HGFA” as used hereinencompasses native sequence polypeptides, polypeptide variants andfragments of a native sequence polypeptide and polypeptide variants(which are further defined herein) that is capable of proHGF cleavage ina manner similar to wild type HGFA. The HGFA polypeptide describedherein may be that which is isolated from a variety of sources, such asfrom human tissue types or from another source, or prepared byrecombinant or synthetic methods. The terms “HGFA”, “HGFA polypeptide”,“HGFA enzyme”, and “HGFA protein” also include variants of a HGFApolypeptide as disclosed herein.

A “native sequence HGFA polypeptide” comprises a polypeptide having thesame amino acid sequence as the corresponding HGFA polypeptide derivedfrom nature (e.g., the sequences depicted in FIG. 7). In one embodiment,a native sequence HGFA polypeptide comprises the amino acid sequence ofSEQ ID NO:1 (see FIG. 7; top sequence). Such native sequence HGFApolypeptide can be isolated from nature or can be produced byrecombinant or synthetic means. The term “native sequence HGFApolypeptide” specifically encompasses naturally-occurring truncated orsecreted forms of the specific HGFA polypeptide (e.g., an extracellulardomain sequence), naturally-occurring variant forms (e.g., alternativelyspliced forms) and naturally-occurring allelic variants of thepolypeptide.

“HGFA polypeptide variant”, or variations thereof, means a HGFApolypeptide, generally an active HGFA polypeptide, as defined hereinhaving at least about 80% amino acid sequence identity with any of thenative sequence HGFA polypeptide sequences as disclosed herein. SuchHGFA polypeptide variants include, for instance, HGFA polypeptideswherein one or more amino acid residues are added, or deleted, at the N-or C-terminus of a native amino acid sequence. Ordinarily, a HGFApolypeptide variant will have at least about 80% amino acid sequenceidentity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% aminoacid sequence identity, to a native sequence HGFA polypeptide sequenceas disclosed herein. Ordinarily, HGFA variant polypeptides are at leastabout 10 amino acids in length, alternatively at least about 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600 aminoacids in length, or more. Optionally, HGFA variant polypeptides willhave no more than one conservative amino acid substitution as comparedto a native HGFA polypeptide sequence, alternatively no more than 2, 3,4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitution as comparedto the native HGFA polypeptide sequence.

“Percent (%) amino acid sequence identity” with respect to a peptide orpolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the specific peptide or polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program ALIGN-2, wherein the complete source code for theALIGN-2 program is provided in Table A below. The ALIGN-2 sequencecomparison computer program was authored by Genentech, Inc. and thesource code shown in Table A below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin FIG. 8 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A.

Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “recombinantvectors”). In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase, or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter synthesis, such as by conjugation with a label. Other types ofmodifications include, for example, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, .alpha.-anomeric sugars, epimeric sugars suchas arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and abasic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), “(O)NR.sub.2 (“amidate”), P(O)R,P(O)OR′, CO or CH.sub.2 (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C.)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat”, and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues may be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence. Unlessindicated otherwise, numbering of all amino acid positions herein isaccording to the Kabat numbering system.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residue in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al. In one embodiment, for the VL, the subgroup is subgroupkappa I as in Kabat et al. In one embodiment, for the VH, the subgroupis subgroup III as in Kabat et al.

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy chain subgroupIII of Kabat et al. In one embodiment, the VH subgroup III consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ IDNO:46)-H1-WVRQAPGKGLEWV (SEQ ID NO:47)-H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:48)-H3-WGQGTLVTVSS (SEQ ID NO:49).

A “VL subgroup I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al. In one embodiment, the VL subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences: DIQMTQSPSSLSASVGDRVTITC- (SEQ ID NO:50)L1-WYQQKPGKAPKLLIY- (SEQ ID NO:51) L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC-(SEQ ID NO:52) L3-FGQGTKVEIK. (SEQ ID NO:53)

The term “hepatocyte growth factor” or “HGF”, as used herein, refers,unless specifically or contextually indicated otherwise, to any nativeor variant (whether naturally occurring or synthetic) HGF polypeptidethat is capable of activating the HGF/c-met signaling pathway underconditions that permit such process to occur. The term “wild type HGF”generally refers to a polypeptide comprising the amino acid sequence ofa naturally occurring HGF protein. Thet term “wild type HGF sequence”generally refers to an amino acid sequence found in a naturallyoccurring HGF.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (for e.g., fulllength or intact monoclonal antibodies), polyclonal antibodies,multivalent antibodies, multispecific antibodies (e.g., bispecificantibodies so long as they exhibit the desired biological activity) andmay also include certain antibody fragments (as described in greaterdetail herein). An antibody can be human, humanized and/or affinitymatured.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion preferably retains at least one, preferably most orall, of the functions normally associated with that portion when presentin an intact antibody. In one embodiment, an antibody fragment comprisesan antigen binding site of the intact antibody and thus retains theability to bind antigen. In another embodiment, an antibody fragment,for example one that comprises the Fc region, retains at least one ofthe biological functions normally associated with the Fc region whenpresent in an intact antibody, such as FcRn binding, antibody half lifemodulation, ADCC function and complement binding. In one embodiment, anantibody fragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For e.g., such an antibodyfragment may comprise on antigen binding arm linked to an Fc sequencecapable of conferring in vivo stability to the fragment.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariabie region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). Preferred affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.Marks et al. Bio/Technology 10:779-783 (1992) describes affinitymaturation by VH and VL domain shuffling. Random mutagenesis of CDRand/or framework residues is described by: Barbas et al. Proc Nat. Acad.Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995);Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J.Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889-896 (1992).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Preferredblocking antibodies or antagonist antibodies substantially or completelyinhibit the biological activity of the antigen.

An “agonist antibody”, as used herein, is an antibody which mimics atleast one of the functional activities of a polypeptide of interest.

A “disorder” is any condition that would benefit from treatment with asubstance/molecule or method of the invention. This includes chronic andacute disorders or diseases including those pathological conditionswhich predispose the mammal to the disorder in question. Non-limitingexamples of disorders to be treated herein include malignant and benigntumors; non-leukemias and lymphoid malignancies; neuronal, glial,astrocytal, hypothalamic and other glandular, macrophagal, epithelial,stromal and blastocoelic disorders; and inflammatory, immunologic andother angiogenesis-related disorders.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer”, “cancerous”, “cellproliferative disorder”, “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include butare not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. More particular examples of such cancers include squamous cellcancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer.

Dysregulation of angiogenesis can lead to many disorders that can betreated by compositions and methods of the invention. These disordersinclude both non-neoplastic and neoplastic conditions. Neoplasticsinclude but are not limited to those described above. Non-neoplasticdisorders include but are not limited to undesired or aberranthypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, psoriaticplaques, sarcoidosis, atherosclerosis, atherosclerotic plaques, diabeticand other proliferative retinopathies including retinopathy ofprematurity, retrolental fibroplasia, neovascular glaucoma, age-relatedmacular degeneration, diabetic macular edema, cornealneovascularization, corneal graft neovascularization, corneal graftrejection, retinal/choroidal neovascularization, neovascularization ofthe angle (rubeosis), ocular neovascular disease, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma,thyroid hyperplasias (including Grave's disease), corneal and othertissue transplantation, chronic inflammation, lung inflammation, acutelung injury/ARDS, sepsis, primary pulmonary hypertension, malignantpulmonary effusions, cerebral edema (e.g., associated with acutestroke/closed head injury/trauma), synovial inflammation, pannusformation in RA, myositis ossificans, hypertropic bone formation,osteoarthritis (OA), refractory ascites, polycystic ovarian disease,endometriosis, 3rd spacing of fluid diseases (pancreatitis, compartmentsyndrome, burns, bowel disease), uterine fibroids, premature labor,chronic inflammation such as IBD (Crohn's disease and ulcerativecolitis), renal allograft rejection, inflammatory bowel disease,nephrotic syndrome, undesired or aberrant tissue mass growth(non-cancer), hemophilic joints, hypertrophic scars, inhibition of hairgrowth, Osler-Weber syndrome, pyogenic granuloma retrolentalfibroplasias, scleroderma, trachoma, vascular adhesions, synovitis,dermatitis, preeclampsia, ascites, pericardial effusion (such as thatassociated with pericarditis), and pleural effusion.

An “autoimmune disease” herein is a non-malignant disease or disorderarising from and directed against an individual's own tissues. Theautoimmune diseases herein specifically exclude malignant or cancerousdiseases or conditions, especially excluding B cell lymphoma, acutelymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hairycell leukemia and chronic myeloblastic leukemia. Examples of autoimmunediseases or disorders include, but are not limited to, inflammatoryresponses such as inflammatory skin diseases including psoriasis anddermatitis (e.g. atopic dermatitis); systemic scleroderma and sclerosis;responses associated with inflammatory bowel disease (such as Crohn'sdisease and ulcerative colitis); respiratory distress syndrome(including adult respiratory distress syndrome; ARDS); dermatitis;meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergicconditions such as eczema and asthma and other conditions involvinginfiltration of T cells and chronic inflammatory responses;atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis;systemic lupus erythematosus (SLE); diabetes mellitus (e.g. Type Idiabetes mellitus or insulin dependent diabetes mellitis); multiplesclerosis; Reynaud's syndrome; autoimmune thyroiditis; allergicencephalomyelitis; Sjorgen's syndrome; juvenile onset diabetes; andimmune responses associated with acute and delayed hypersensitivitymediated by cytokines and T-lymphocytes typically found in tuberculosis,sarcoidosis, polymyositis, granulomatosis and vasculitis; perniciousanemia (Addison's disease); diseases involving leukocyte diapedesis;central nervous system (CNS) inflammatory disorder; multiple organinjury syndrome; hemolytic anemia (including, but not limited tocryoglobinemia or Coombs positive anemia); myasthenia gravis;antigen-antibody complex mediated diseases; anti-glomerular basementmembrane disease; antiphospholipid syndrome; allergic neuritis; Graves'disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous;pemphigus; autoimmune polyendocrinopathies; Reiter's disease; stiff-mansyndrome; Behcet disease; giant cell arteritis; immune complexnephritis; IgA nephropathy; IgM polyneuropathies; immunethrombocytopenic purpura (ITP) or autoimmune thrombocytopenia etc.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing metastasis, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis. In some embodiments, antibodies of theinvention are used to delay development of a disease or disorder.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A “therapeutically effective amount” of a substance/molecule of theinvention, agonist or antagonist may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the substance/molecule, agonist or antagonist to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of thesubstance/molecule, agonist or antagonist are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typicallybut not necessarily, since a prophylactic dose is used in subjects priorto or at an earlier stage of disease, the prophylactically effectiveamount will be less than the therapeutically effective amount.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, and the variousantitumor or anticancer agents disclosed below. Other cytotoxic agentsare described below. A tumoricidal agent causes destruction of tumorcells.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g.,Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HClliposome injection (DOXIL®) and deoxydoxorubicin), epirubicin,esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C,mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin,puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such asmethotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine(XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acidanalogues such as denopterin, methotrexate, pteropterin, trimetrexate;purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine,thioguanine; pyrimidine analogs such as ancitabine, azacitidine,6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,enocitabine, floxuridine; androgens such as calusterone, dromostanolonepropionate, epitiostanol, mepitiostane, testolactone; anti-adrenals suchas aminoglutethimide, mitotane, trilostane; folic acid replenisher suchas frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate;etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine;maytansinoids such as maytansine and ansamitocins; mitoguazone;mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet;pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK®polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane;rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®,FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol;pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, e.g.,paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation ofpaclitaxel (ABRAXANE™), and doxetaxel (TAXOTERE®); chloranbucil;6-thioguanine; mercaptopurine; methotrexate; platinum analogs such ascisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide(VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin;leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate;daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000;difluorometlhylornithine (DMFO); retinoids such as retinoic acid;pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),raloxifene (EVISTA®), droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and toremifene (FARESTON®);anti-progesterones; estrogen receptor down-regulators (ERDs); estrogenreceptor antagonists such as fulvestrant (FASLODEX®); agents thatfunction to suppress or shut down the ovaries, for example, leutinizinghormone-releasing hormone (LHRH) agonists such as leuprolide acetate(LUPRON® and ELIGARD®), goserelin acetate, buserelin acetate andtripterelin; other anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrolacetate (MEGASE®), exemestane (AROMASIN®), formestanie, fadrozole,vorozole (RIVISOR®), letrozole (FEMARA®), and anastrozole (ARIMIDEX®).In addition, such definition of chemotherapeutic agents includesbisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®),alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), orrisedronate (ACTONEL®); as well as troxacitabine (a 1,3-dioxolanenucleoside cytosine analog); antisense oligonucleotides, particularlythose that inhibit expression of genes in signaling pathways implicatedin abherant cell proliferation, such as, for example, PKC-alpha, Raf,H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such asTHERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1inhibitor (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); lapatinibditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-moleculeinhibitor also known as GW572016); COX-2 inhibitors such as celecoxib(CELEBREX®; 4-(5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell whose growth is dependentupon HGF/c-met activation either in vitro or in vivo. Thus, the growthinhibitory agent may be one which significantly reduces the percentageof HGF/c-met-dependent cells in S phase. Examples of growth inhibitoryagents include agents that block cell cycle progression (at a placeother than S phase), such as agents that induce G1 arrest and M-phasearrest. Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (W B Saunders:Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel anddocetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

“Doxorubicin” is an anthracycline antibiotic. The full chemical name ofdoxorubicin is(8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.

Vectors Host Cells and Recombinant Methods

For recombinant production of an antibody of the invention, the nucleicacid encoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The choice ofvector depends in part on the host cell to be used. Generally, preferredhost cells are of either prokaryotic or eukaryotic (generally mammalian)origin.

Generating Antibodies Using Prokaryotic Host Cells:

Vector Construction

Polynucleotide sequences encoding polypeptide components of the antibodyof the invention can be obtained using standard recombinant techniques.Desired polynucleotide sequences may be isolated and sequenced fromantibody producing cells such as hybridoma cells. Alternatively,polynucleotides can be synthesized using nucleotide synthesizer or PCRtechniques. Once obtained, sequences encoding the polypeptides areinserted into a recombinant vector capable of replicating and expressingheterologous polynucleotides in prokaryotic hosts. Many vectors that areavailable and known in the art can be used for the purpose of thepresent invention. Selection of an appropriate vector will depend mainlyon the size of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. Inducible promoter isa promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g. the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the signal sequences native to the heterologouspolypeptides, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group consisting of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of theinvention, the signal sequences used in both cistrons of the expressionsystem are STII signal sequences or variants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. coli trxB⁻strains) provide cytoplasm conditions that are favorable for disulfidebond formation, thereby permitting proper folding and assembly ofexpressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

The present invention provides an expression system in which thequantitative ratio of expressed polypeptide components can be modulatedin order to maximize the yield of secreted and properly assembledantibodies of the invention. Such modulation is accomplished at least inpart by simultaneously modulating translational strengths for thepolypeptide components.

One technique for modulating translational strength is disclosed inSimmons et al., U.S. Pat. No. 5,840,523. It utilizes variants of thetranslational initiation region (TIR) within a cistron. For a given TIR,a series of amino acid or nucleic acid sequence variants can be createdwith a range of translational strengths, thereby providing a convenientmeans by which to adjust this factor for the desired expression level ofthe specific chain. TIR variants can be generated by conventionalmutagenesis techniques that result in codon changes which can alter theamino acid sequence, although silent changes in the nucleotide sequenceare preferred. Alterations in the TIR can include, for example,alterations in the number or spacing of Shine-Dalgarno sequences, alongwith alterations in the signal sequence. One method for generatingmutant signal sequences is the generation of a “codon bank” at thebeginning of a coding sequence that does not change the amino acidsequence of the signal sequence (i.e., the changes are silent). This canbe accomplished by changing the third nucleotide position of each codon;additionally, some amino acids, such as leucine, serine, and arginine,have multiple first and second positions that can add complexity inmaking the bank. This method of mutagenesis is described in detail inYansura et al. (1992) METHODS: A Companion to Methods in Enzymol.4:151-158.

Preferably, a set of vectors is generated with a range of TIR strengthsfor each cistron therein. This limited set provides a comparison ofexpression levels of each chain as well as the yield of the desiredantibody products under various TIR strength combinations. TIR strengthscan be determined by quantifying the expression level of a reporter geneas described in detail in Simmons et al. U.S. Pat. No. 5,840,523. Basedon the translational strength comparison, the desired individual TIRsare selected to be combined in the expression vector constructs of theinvention.

Prokaryotic host cells suitable for expressing antibodies of theinvention include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. coli), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negativecells are used. In one embodiment, E. coli cells are used as hosts forthe invention. Examples of E. coli strains include strain W3110(Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.:American Society for Microbiology, 1987), pp. 1190-1219; ATCC DepositNo. 27,325) and derivatives thereof, including strain 33D3 havinggenotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41kan^(R) (U.S. Pat. No. 5,639,635). Other strains and derivativesthereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli _(λ) 1776(ATCC 31,537) and E. coli RV308 (ATCC 31,608) are also suitable. Theseexamples are illustrative rather than limiting. Methods for constructingderivatives of any of the above-mentioned bacteria having definedgenotypes are known in the art and described in, for example, Bass etal., Proteins, 8:309-314 (1990). It is generally necessary to select theappropriate bacteria taking into consideration replicability of thereplicon in the cells of a bacterium. For example, E. coli, Serratia, orSalmonella species can be suitably used as the host when well knownplasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supplythe replicon. Typically the host cell should secrete minimal amounts ofproteolytic enzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

Antibody Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. Preferably, thephosphate-limiting medium is the C.R.A.P medium (see, for e.g., Simmonset al., J. Immunol. Methods (2002), 263:133-147). A variety of otherinducers may be used, according to the vector construct employed, as isknown in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay.

In one aspect of the invention, antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, preferably about 1,000 to 100,000 liters of capacity. Thesefermentors use agitator impellers to distribute oxygen and nutrients,especially glucose (the preferred carbon/energy source). Small scalefermentation refers generally to fermentation in a fermentor that is nomore than approximately 100 liters in volumetric capacity, and can rangefrom about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD₅₅₀ of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain-host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease III, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention.

Antibody Purification

In one embodiment, the antibody protein produced herein is furtherpurified to obtain preparations that are substantially homogeneous forfurther assays and uses. Standard protein purification methods known inthe art can be employed. The following procedures are exemplary ofsuitable purification procedures: fractionation on immunoaffinity orion-exchange columns, ethanol precipitation, reverse phase HPLC,chromatography on silica or on a cation-exchange resin such as DEAE,chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gelfiltration using, for example, Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the full length antibody products of theinvention. Protein A is a 41 kD cell wall protein from Staphylococcusaureas which binds with a high affinity to the Fc region of antibodies.Lindmark et al (1983) J. Immunol. Meth. 62:1-13. The solid phase towhich Protein A is immobilized is preferably a column comprising a glassor silica surface, more preferably a controlled pore glass column or asilicic acid column. In some applications, the column has been coatedwith a reagent, such as glycerol, in an attempt to prevent nonspecificadherence of contaminants.

As the first step of purification, the preparation derived from the cellculture as described above is applied onto the Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase is then washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

Generating Antibodies Using Eukaryotic Host Cells:

The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence.

(i) Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(ii) Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCCCRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodypolypeptide nucleic acid. Promoter sequences are known for eukaryotes.Virtually alleukaryotic genes have an AT-rich region locatedapproximately 25 to 30 bases upstream from the site where transcriptionis initiated. Another sequence found 70 to 80 bases upstream from thestart of transcription of many genes is a CNCAAT region where N may beany nucleotide. At the 3′ end of most eukaryotic genes is an AATAAAsequence that may be the signal for addition of the poly A tail to the3′ end of the coding sequence. All of these sequences are suitablyinserted into eukaryotic expression vectors.

Antibody polypeptide transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(v) Enhancer Element Component

Transcription of DNA encoding the antibody polypeptide of this inventionby higher eukaryotes is often increased by inserting an enhancersequence into the vector. Many enhancer sequences are now known frommammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theantibody polypeptide-encoding sequence, but is preferably located at asite 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells will typically alsocontain sequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(ix) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, are removed, for example, bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Activity Assays

The antibodies of the present invention can be characterized for theirphysical/chemical properties and biological functions by various assaysknown in the art.

The purified immunoglobulins can be further characterized by a series ofassays including, but not limited to, N-terminal sequencing, amino acidanalysis, non-denaturing size exclusion high pressure liquidchromatography (HPLC), mass spectrometry, ion exchange chromatographyand papain digestion.

In certain embodiments of the invention, the immunoglobulins producedherein are analyzed for their biological activity. In some embodiments,the immunoglobulins of the present invention are tested for theirantigen binding activity. The antigen binding assays that are known inthe art and can be used herein include without limitation any direct orcompetitive binding assays using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, fluorescent immunoassays, andprotein A immunoassays. An illustrative antigen binding assay isprovided below in the Examples section.

In one embodiment, the present invention contemplates an alteredantibody that possesses some but not all effector functions, which makeit a desired candidate for many applications in which the half life ofthe antibody in vivo is important yet certain effector functions (suchas complement and ADCC) are unnecessary or deleterious. In certainembodiments, the Fc activities of the produced immunoglobulin aremeasured to ensure that only the desired properties are maintained. Invitro and/or in vivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Anexample of an in vitro assay to assess ADCC activity of a molecule ofinterest is described in U.S. Pat. No. 5,500,362 or 5,821,337. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.PNAS (USA) 95:652-656 (1998). C1q binding assays may also be carried outto confirm that the antibody is unable to bind C1q and hence lacks CDCactivity. To assess complement activation, a CDC assay, for e.g. asdescribed in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996),may be performed. FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art.

Humanized Antibodies

The present invention encompasses humanized antibodies. Various methodsfor humanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source which is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536), by substitutinghypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some hypervariable region residuesand possibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody (Sims et al. (1993) J.Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285;Presta et al. (1993) J. Immunol., 151:2623.

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to one method, humanized antibodies areprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from therecipient and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the hypervariable region residues are directly andmost substantially involved in influencing antigen binding.

Antibody Variants

In one aspect, the invention provides antibody comprising modificationsin the interface of Fc polypeptides comprising the Fc region, whereinthe modifications facilitate and/or promote heterodimerization. Thesemodifications comprise introduction of a protuberance into a first Fcpolypeptide and a cavity into a second Fc polypeptide, wherein theprotuberance is positionable in the cavity so as to promote complexingof the first and second Fc polypeptides. Methods of generatingantibodies with these modifications are known in the art, for e.g., asdescribed in U.S. Pat. No. 5,731,168.

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodyare prepared by introducing appropriate nucleotide changes into theantibody nucleic acid, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of, residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution ismade to arrive at the final construct, provided that the final constructpossesses the desired characteristics. The amino acid alterations may beintroduced in the subject antibody amino acid sequence at the time thatsequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressedimmunoglobulins are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g. for ADEPF) or a polypeptide which increases the serumhalf-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin Table 1 under the heading of “preferred substitutions”. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table 1,or as further described below in reference to amino acid classes, may beintroduced and the products screened. TABLE 1 Original ExemplaryPreferred Residue Substitutions Substitutions Ala (A) Val; Leu; Ile ValArg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D)Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; GlnAsp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine; Ile; Val; Ile Met;Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F)Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T)Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V)Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Amino acids maybe grouped according to similarities in the properties of their sidechains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75,Worth Publishers, New York (1975)):

(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp(W), Met (M)

(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn(N), Gln (O)

(3) acidic: Asp (D), Glu (E)

(4) basic: Lys (K), Arg (R), His (H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have improved biological properties relative tothe parent antibody from which they are generated. A convenient way forgenerating such substitutional variants involves affinity maturationusing phage display. Briefly, several hypervariable region sites (e.g.6-7 sites) are mutated to generate all possible amino acid substitutionsat each site. The antibodies thus generated are displayed fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and antigen. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of the immunoglobulin polypeptides of the invention,thereby generating a Fc region variant. The Fc region variant maycomprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 orIgG4 Fc region) comprising an amino acid modification (e.g. asubstitution) at one or more amino acid positions including that of ahinge cysteine.

In accordance with this description and the teachings of the art, it iscontemplated that in some embodiments, an antibody used in methods ofthe invention may comprise one or more alterations as compared to thewild type counterpart antibody, for e.g. in the Fc region. Theseantibodies would nonetheless retain substantially the samecharacteristics required for therapeutic utility as compared to theirwild type counterpart. For e.g., it is thought that certain alterationscan be made in the Fc region that would result in altered (i.e., eitherimproved or diminished) C1q binding and/or Complement DependentCytotoxicity (CDC), for e.g., as described in WO99/51642. See alsoDuncan & Winter Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S.Pat. No. 5,624,821; and WO94/29351 concerning other examples of Fcregion variants.

Immunoconjugates

The invention also pertains to immunoconjugates, or antibody-drugconjugates (ADC), comprising an antibody conjugated to a cytotoxic agentsuch as a chemotherapeutic agent, a drug, a growth inhibitory agent, atoxin (e.g., an enzymatically active toxin of bacterial, fungal, plant,or animal origin, or fragments thereof), or a radioactive isotope (i.e.,a radioconjugate).

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, i.e. drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos (1999) Anticancer Research19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev.26:151-172; U.S. Pat. No. 4,975,278) theoretically allows targeteddelivery of the drug moiety to tumors, and intracellular accumulationtherein, where systemic administration of these unconjugated drug agentsmay result in unacceptable levels of toxicity to normal cells as well asthe tumor cells sought to be eliminated (Baldwin et al., (1986) Lancetpp. (Mar. 15, 1986):603-05; Thorpe, (1985) “Antibody Carriers OfCytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies'84: Biological And Clinical Applications, A. Pinchera et al. (ed.s),pp. 475-506). Maximal efficacy with minimal toxicity is sought thereby.Both polyclonal antibodies and monoclonal antibodies have been reportedas useful in these strategies (Rowland et al., (1986) Cancer Immunol.Immunother., 21:183-87). Drugs used in these methods include daunomycin,doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra).Toxins used in antibody-toxin conjugates include bacterial toxins suchas diphtheria toxin, plant toxins such as ricin, small molecule toxinssuch as geldanamycin (Mandler et al (2000) Jour. of the Nat. CancerInst. 92(19): 1573-1581; Mandler et al (2000) Bioorganic & Med. Chem.Letters 10: 1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Rinman et al (1993) Cancer Res. 53:3336-3342). Thetoxins may effect their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.Some cytotoxic drugs tend to be inactive or less active when conjugatedto large antibodies or protein receptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and ¹¹¹In or ⁹⁰Y radioisotope bound by a thiourealinker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al(2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Clin.Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cellnon-Hodgkin's Lymphoma (NHL), administration results in severe andprolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin,Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33antibody linked to calicheamicin, was approved in 2000 for the treatmentof acute myeloid leukemia by injection (Drugs of the Future (2000)25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040;5,693,762; 5,739,116; 5,767,285; 5,773,001). Cantuzumab mertansine(Immunogen, Inc.), an antibody drug conjugate composed of the huC242antibody linked via the disulfide linker SPP to the maytansinoid drugmoiety, DM1, is advancing into Phase II trials for the treatment ofcancers that express CanAg, such as colon, pancreatic, gastric, andothers. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), anantibody drug conjugate composed of the anti-prostate specific membraneantigen (PSMA) monoclonal antibody linked to the maytansinoid drugmoiety, DM1, is under development for the potential treatment ofprostate tumors. The auristatin peptides, auristatin E (AE) andmonomethylauristatin (MMAE), synthetic analogs of dolastatin, wereconjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Yon carcinomas) and cAC10 (specific to CD30 on hematologicalmalignancies) (Doronina et al (2003) Nature Biotechnology 21(7):778-784)and are under therapeutic development.

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made usinga variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such asbis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, a trichothecene, and CC1065, and thederivatives of these toxins that have toxin activity, are alsocontemplated herein.

Maytansine and Maytansinoids

In one embodiment, an antibody (full length or fragments) of theinvention is conjugated to one or more maytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533, the disclosures of which are hereby expressly incorporated byreference.

Maytansinoid-Antibody Conjugates

In an attempt to improve their therapeutic index, maytansine andmaytansinoids have been conjugated to antibodies specifically binding totumor cell antigens. Immunoconjugates containing maytansinoids and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1, the disclosures of whichare hereby expressly incorporated by reference. Liu et al., Proc. Natl.Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprisinga maytansinoid designated DM1 linked to the monoclonal antibody C242directed against human colorectal cancer. The conjugate was found to behighly cytotoxic towards cultured colon cancer cells, and showedantitumor activity in an in vivo tumor growth assay. Chari et al.,Cancer Research 52:127-131 (1992) describe immunoconjugates in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA.1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×10⁵ HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansinoid drug, whichcould be increased by increasing the number of maytansinoid moleculesper antibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Antibody-Maytansinoid Conjugates (Immunoconjugates)

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. An average of 3-4 maytansinoid molecules conjugated perantibody molecule has shown efficacy in enhancing cytotoxicity of targetcells without negatively affecting the function or solubility of theantibody, although even one molecule of toxin/antibody would be expectedto enhance cytotoxicity over the use of naked antibody. Maytansinoidsare well known in the art and can be synthesized by known techniques orisolated from natural sources. Suitable maytansinoids are disclosed, forexample, in U.S. Pat. No. 5,208,020 and in the other patents andnonpatent publications referred to hereinabove. Preferred maytansinoidsare maytansinol and maytansinol analogues modified in the aromatic ringor at other positions of the maytansinol molecule, such as variousmaytansinol esters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, andChari et al., Cancer Research 52:127-131 (1992). The linking groupsinclude disulfide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups, or esterase labile groups,as disclosed in the above-identified patents, disulfide and thioethergroups being preferred.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas his (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 [1978]) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

Calicheamicin

Another immunoconjugate of interest comprises an antibody conjugated toone or more calicheamicin molecules. The calicheamicin family ofantibiotics are capable of producing double-stranded DNA breaks atsub-picomolar concentrations. For the preparation of conjugates of thecalicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586,5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all toAmerican Cyanamid Company). Structural analogues of calicheamicin whichmay be used include, but are not limited to, γ₁ ^(I), α₂ ^(I), α₃ ^(I),N-acetyl-γ₁ ^(I), PSAG and θ^(I) ₁ (Hinman et al., Cancer Research53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998)and the aforementioned U.S. patents to American Cyanamid). Anotheranti-tumor drug that the antibody can be conjugated is QFA which is anantifolate. Both calicheamicin and QFA have intracellular sites ofaction and do not readily cross the plasma membrane. Therefore, cellularuptake of these agents through antibody mediated internalization greatlyenhances their cytotoxic effects.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for exampletc^(99m) or I¹²³, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS,HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, andsulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which arecommercially available (e.g., from Pierce Biotechnology, Inc., Rockford,Ill., U.S.A). See pages 467-498, 2003-2004 Applications Handbook andCatalog.

Preparation of Antibody Drug Conjugates

In the antibody drug conjugates (ADC) of the invention, an antibody (Ab)is conjugated to one or more drug moieties (D), e.g. about 1 to about 20drug moieties per antibody, through a linker (L). The ADC of Formula Imay be prepared by several routes, employing organic chemistryreactions, conditions, and reagents known to those skilled in the art,including: (1) reaction of a nucleophilic group of an antibody with abivalent linker reagent, to form Ab-L, via a covalent bond, followed byreaction with a drug moiety D; and (2) reaction of a nucleophilic groupof a drug moiety with a bivalent linker reagent, to form D-L, via acovalent bond, followed by reaction with the nucleophilic group of anantibody.Ab-(L-D)_(p)  I

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol.

Antibody drug conjugates of the invention may also be produced bymodification of the antibody to introduce electrophilic moieties, whichcan react with nucleophilic subsituents on the linker reagent or drug.The sugars of glycosylated antibodies may be oxidized, e.g. withperiodate oxidizing reagents, to form aldehyde or ketone groups whichmay react with the amine group of linker reagents or drug moieties. Theresulting imine Schiff base groups may form a stable linkage, or may bereduced, e.g. by borohydride reagents to form stable amine linkages. Inone embodiment, reaction of the carbohydrate portion of a glycosylatedantibody with either glactose oxidase or sodium meta-periodate may yieldcarbonyl (aldehyde and ketone) groups in the protein that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, proteins containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Suchaldehyde can be reacted with a drug moiety or linker nucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin)which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

Antibody Derivatives

The antibodies of the present invention can be further modified tocontain additional nonproteinaceous moieties that are known in the artand readily available. Preferably, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymers are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

Pharmaceutical Formulations

Therapeutic formulations comprising an antibody of the invention areprepared for storage by mixing the antibody having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of aqueous solutions, lyophilized or otherdried formulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, histidine and other organicacids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the immunoglobulin of the invention,which matrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPO™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated immunoglobulins remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

In certain embodiments, an immunoconjugate comprising an antibodyconjugated with a cytotoxic agent is administered to the patient. Insome embodiments, the immunoconjugate and/or antigen to which it isbound is/are internalized by the cell, resulting in increasedtherapeutic efficacy of the immunoconjugate in killing the target cellto which it binds. In one embodiment, the cytotoxic agent targets orinterferes with nucleic acid in the target cell. Examples of suchcytotoxic agents include any of the chemotherapeutic agents noted herein(such as a maytansinoid or a calicheamicin), a radioactive isotope, or aribonuclease or a DNA endonuclease.

Antibodies of the invention can be used either alone or in combinationwith other compositions in a therapy. For instance, an antibody of theinvention may be co-administered with another antibody, chemotherapeuticagent(s) (including cocktails of chemotherapeutic agents), othercytotoxic agent(s), anti-angiogenic agent(s), cytokines, and/or growthinhibitory agent(s). Where an antibody of the invention inhibits tumorgrowth, it may be particularly desirable to combine it with one or moreother therapeutic agent(s) which also inhibits tumor growth. Forinstance, an antibody of the invention may be combined with anti-VEGFantibodies blocking VEGF activities and/or anti-ErbB antibodies (e.g.HERCEPTIN® anti-HER2 antibody) in a treatment of metastatic breastcancer. Alternatively, or additionally, the patient may receive combinedradiation therapy (e.g. external beam irradiation or therapy with aradioactive labeled agent, such as an antibody). Such combined therapiesnoted above include combined administration (where the two or moreagents are included in the same or separate formulations), and separateadministration, in which case, administration of the antibody of theinvention can occur prior to, and/or following, administration of theadjunct therapy or therapies.

The antibody of the invention (and adjunct therapeutic agent) is/areadministered by any suitable means, including parenteral, subcutaneous,intraperitoneal, intrapulmonary, and intranasal, and, if desired forlocal treatment, intralesional administration. Parenteral infusionsinclude intramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antibody is suitablyadministered by pulse infusion, particularly with declining doses of theantibody. Dosing can be by any suitable route, for e.g. by injections,such as intravenous or subcutaneous injections, depending in part onwhether the administration is brief or chronic.

The antibody composition of the invention will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount ofantibodies of the invention present in the formulation, the type ofdisorder or treatment, and other factors discussed above. These aregenerally used in the same dosages and with administration routes asused hereinbefore or about from 1 to 99% of the heretofore employeddosages.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with otheragents such as chemotherapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofantibody is an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment is sustained until a desiredsuppression of disease symptoms occurs. One exemplary dosage of theantibody would be in the range from about 0.05 mg/kg to about 10 mg/kg.Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10mg/kg (or any combination thereof) may be administered to the patient.Such doses may be administered intermittently, e.g. every week or everythree weeks (e.g. such that the patient receives from about two to abouttwenty, e.g. about six doses of the antibody). An initial higher loadingdose, followed by one or more lower doses may be administered. Anexemplary dosing regimen comprises administering an initial loading doseof about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kgof the antibody. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is by itself or when combined with another composition effectivefor treating, preventing and/or diagnosing the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is anantibody of the invention. The label or package insert indicates thatthe composition is used for treating the condition of choice, such ascancer. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic agent. The article of manufacture in this embodimentof the invention may further comprise a package insert indicating thatthe first and second antibody compositions can be used to treat aparticular condition, for e.g. cancer. Alternatively, or additionally,the article of manufacture may further comprise a second (or third)container comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

The following are examples of the methods and compositions of theinvention. It is understood that various other embodiments may bepracticed, given the general description provided above.

EXAMPLES

Materials & Methods

Reagents

Corn trypsin inhibitor was from Haematologic Technologies (EssexJunction, Vt.) and the chromogenic substrate for HGFA, Spectrozyme®fVIIa, was from American Diagnostica (Stamford, Conn.). Soluble HAI-1B(sHAI-1B) was expressed in Chinese Hamster Ovary cells and purified aspreviously described (1). The Kunitz domain inhibitor IV-49C waspreviously described (2) (Genentech, Inc., South San Francisco). Humanrecombinant HGFA (HGFA) was expressed in a baculovirus expression systemas previously described (1).

ProHGF Activation Assays

ProHGF activation assays and proHGF labeling with Iodogen were carriedout as previously described (1,3). Briefly, HGFA was preincubated withanti-HGFA antibodies or sHAI-1B in HNC buffer (20 mM Hepes, pH 7.5, 150mM NaCl, 5 mM CaCl₂) for 15 min at room temperature, after which¹²⁵I-labeled proHGF in HNC buffer was added and incubated for 4 hrs at37° C. The reactant concentrations in the final mixture were as follows:2 nM HGFA, 0.05 mg/ml ¹²⁵I-labeled proHGF, 0.1 mg/ml anti-HGFAantibodies, 1 μM sHAI-1B. After 4 hrs aliquots were removed and added tosample buffer (Bio-Rad Laboratories, Hercules, Calif.) with reducingagent dithiotreitol (BIO-Rad). After a brief heating, samples (approx.10⁶ cpm/lane) were loaded onto a 4-20% gradient polyacrylamide gel(Invitrogen Corp., Carlsbad, Calif.). After electrophoresis, the driedgels were exposed on x-ray films (X-OMAT AR, Eastman Kodak Company,Rochester, N.Y.) for 10-20 min. Films were developed (Kodak M35A X-OMATProcessor), scanned (Umax S-12, Umax Data Systems, Inc., Fremont,Calif.) and further processed with Adobe V.6.0 Photoshop software (AdobeSystems Inc., San Jose, Calif.).

BIAcore Experiments

Binding affinities of anti-HGFA antibodies to HGFA were determined bysurface plasmon resonance measurements on a BIAcore 3000 instrument(Biacore, Inc.) The reformatted full length anti-HGFA IgG1 wasimmobilized at a density of 300 resonance units (RU) on the flow cellsof a Pioneer CM5 sensor chip. Immobilization was achieved by randomcoupling through amino groups using a protocol provided by themanufacturer. Sensorgrams were recorded for binding of HGFA to thesesurfaces by injection of a series of solutions ranging from 1 μM to 8 nMin 2-fold increments. The signal from the reference cell was subtractedfrom the observed sensorgram. Kinetic constants were calculated bynonlinear regression analysis of the data according to a 1:1 Languirbinding model using software supplied by the manufacturer. Incompetition experiments, HGFA (70 nM) was preincubated with variousconcentrations of sHAI-1B (4 nM-300 nM) or IV49C (11 nM-300 nM) or asmall molecule HGFA active site binder (220 nM-10 uM). After incubationfor 60 min at room temperature, the enzyme-inhibitor mixture wasinjected into the flow cells and sensorgrams recorded.

HGFA Enzyme Inhibition Assay

The antibodies or sHAI-1B were incubated with HGFA (final concentration5 nM) in HBSA buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 0.5 mg/ml BSA, 5mM CaCl₂) for 20 min at room temperature. Spectrozyme® fvIIa (200 μMfinal conc., K_(M)=200 μM) was added and the linear rates of theincrease in absorbance at 405 nm measured on a kinetic microplate reader(Molecular Devices, Sunnyvale, Calif.). Inhibition of enzyme activitywas expressed as fractional activity (v_(i)/v_(o)) of uninhibitedactivity.

Results & Discussion

Identification of Anti-HGFA Antibodies by Phage Display

One method of identifying antibodies is through the use of a phageantibody library. See, for example, Lee et al. (4). To identifyantibodies against HGFA, we carried out four rounds of panning using apreviously reported human synthetic phagemid antibody library (a F(ab′)₂library). Plates were coated with 5 μg/well of HGFA. We increasedstringency of washing after each round, from 10-40 times of washes. Weobserved enrichment after three rounds of panning. After four rounds ofpanning, 95 clones were picked for ELISA assays. After sequencing, 67unique clones were found to specifically bind to HGFA. After spotcompetition ELISA, 24 clones were further characterized using purifiedphage to measure IC₅₀ values, which were determined using a standardphage competition ELISA. 14 unique clones with IC₅₀ values <100 nM weresub-cloned into PRK-human IgG1 vector. CDR sequences for these clonesare listed in FIG. 1. Heavy and light chains (from the humanized 4D5antibody as described in Lee et al. (4)) of anti-HGFA clones wereco-transfected into mammalian 293 cells. After one week, the serum-freesupernatants were harvested and the antibodies purified using protein Aaffinity chromatography.

Inhibition of HGFA Enzymatic Activity by Full-Length Anti-HGFAAntibodies

The selected antibodies were reformatted as full-length antibodies (IgG)by standard recombinant techniques. These full length antibodies wereexamined in a macromolecular substrate activation assay using¹²⁵I-labelled proHGF. During the 4 hr experiment, HGFA completelyconverted proHGF into 2-chain HGF, and this reaction could be inhibitedby 1 μM sHAI-1B (FIG. 2A) consistent with previous reports (1). With theexception of antibody #49 (FIG. 1), all tested anti-HGFA antibodies atthe tested concentration of 0.67 μM significantly inhibited proHGFconversion (FIG. 2). Additional experiments showed that #58 inhibitedproHGF conversion at concentrations as low as 0.03 μM (FIG. 3).Consistent with these results, antibody #58 most potently inhibited HGFAenzymatic activity towards the small synthetic substrate Spectrozyme®fVIIa, having an IC₅₀ of 1.3 nM, whereas antibody #49 did not inhibit at500 nM (FIG. 8). Furthermore, in agreement with their relatively weakerinhibitory activities in proHGF activation assays, the antibodies #39,#86, #90 and #95 had comparably weaker activities in the chromogenicsubstrate assay, having IC₅₀>500 nM (FIG. 8). The 3 antibodies #42, #61,and #74 also showed relatively weak inhibition (IC₅₀>500 nM) despitealmost complete inhibition of proHGF conversion at 0.67 μM (FIG. 8).Interestingly, antibody #75 displayed unusual inhibition kinetics inthat its inhibitory activity reached a plateau at about 70% inhibitionas compared to the complete inhibition achieved by antibody #58 (FIG.4).

Inhibitory Mechanisms of Antibodies #75 and #58

In light of the complete inhibition of macromolecular substrateprocessing by antibody #75, its inability to completely neutralize HGFAenzymatic activity towards the small synthetic substrate suggested thatantibody #75 binds to a functionally important HGFA region locatedoutside, or in proximity to, the active site. In contrast, antibody #58strongly inhibited both the macromolecular and small substrateprocessing by HGFA. To gain more detailed insight into the antibodies'inhibitory mechanisms, competition binding studies with various knownactive site inhibitors were carried out. The three HGFA active siteinhibitors used were the previously described bi-Kunitz domain inhibitorsHAI-1B (1), the single Kunitz domain inhibitor IV-49C (2) and the smallmolecule HGFA active site binder. IV-49C is a 62 amino acid Kunitzdomain derived from Alzheimer's β-protein precursor inhibitor (APPI) andis a specific inhibitor of the tissue factor/factor VIIa complex (2). Wefound that IV-49C is also a potent inhibitor of HGFA enzymatic activity,having an IC₅₀ of 0.079 μM, whereas the small molecule HGFA active sitebinder inhibited with an IC₅₀ of 0.8 μM (K_(i)=0.4 μM) as shown in FIG.5.

The K_(D) of HGFA to immobilized antibody #58 was 1.3 nM (FIG. 9),similar to the affinity determined by amidolytic assays (FIG. 8).BIAcore measurements showed that sHAI-1B, IV-49C and the small moleculeHGFA active site binder inhibited HGFA binding to #58. This suggestedthat #58 either binds directly to the active site of HGFA or exertsallosteric influences on the active site.

Antibody #75 had weaker binding to HGFA (FIG. 6E; FIG. 9) than #58.Moreover, the small molecule HGFA active site binder had no effect onHGFA binding to antibody #75, indicating that antibody #75 does not bindto the ‘core’ region of the active site. Interestingly, antibody #75partially inhibited HGFA amidolytic activity, suggesting that eventhough the #75 epitope lies outside the active site, there must be amolecular linkage between these two sites. This would explain thepartial effects of sHAI-1B and IV49C on antibody #75 binding (FIG. 9;FIG. 6F,G).

Similar to antibody #75, the two antibodies #74 and #61 also bound toHGFA in the presence of the small molecule active site binder (FIG. 9),while the Kunitz domain inhibitors interfered with HGFA binding. Theseresults suggested that the epitopes of #74 and #61 lie outside theactive site of HGFA. It is conceivable that the antibodies #61, #74 and#75 bind to an HGFA exosite region that is important for macromolecularsubstrate interaction or that they allosterically influence theconformation of the active site region. In the structurally relatedserine protease factor VIIa an important exosite is located between theactive site and the calcium binding loop (5). Antibodies as well aspeptides which bind to the factor VIIa exosite are potent inhibitors ofmacromolecular substrate processing (6,7). For instance, binding of thepeptidic inhibitor E76 effects conformational changes in one of the‘activation domain’ loops thereby disrupting a substrate interactionsite (7). In addition, these changes induce allosteric effects at theactive site, which explains the observation that E-76 peptide inhibitsamidolytic activity despite binding outside the active site region (7).

Additional competition binding experiments with biotinylated antibodies#75 and #58 indicated that #75 and #58 have overlapping epitopes on HGFA(data not shown). Enzyme kinetic studies further demonstrated that #58is a competitive inhibitor and that #75 is a partial competitiveinhibitor (i.e. simple intersecting hyperbolic competitive inhibitor)(data not shown). Together, these results suggest that both antibodiesbind outside the HGFA active site and that they are allostericinhibitors of HGFA enzymatic activity.

PARTIAL LIST OF REFERENCES

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1. An isolated antibody that binds human hepatocyte growth factoractivator (HGFA).
 2. The antibody of claim 1, wherein the antibody bindsto an active site of HGFA.
 3. The antibody of claim 1, wherein theantibody binds to HGFA at a position other than an active site of HGFA.4. The antibody of claim 1 which is a blocking antibody.
 5. The antibodyof claim 4 which blocks HGFA proteolytic activity.
 6. The antibody ofclaim 5 which blocks HGFA proteolysis of single chain HGF.
 7. Theantibody of claim 1 which inhibits HGF/c-met signaling.
 8. The antibodyof claim 7 which inhibits cell proliferation.
 9. The antibody of claim 7which inhibits angiogenesis.
 10. The antibody of claim 1, wherein theantibody is an allosteric inhibitor of HGFA enzymatic activity.
 11. Theantibody of claim 1, wherein the antibody is a complete or partialcompetitive inhibitor of HGFA enzymatic activity.
 12. A method treatinga disease associated with dysregulation of HGF/c-met signaling in asubject, comprising administering to the subject an effective amount ofan antibody that binds HGFA in accordance with any of the precedingclaims.
 13. The method of claim 10 wherein the disease is cancer. 14.The method of claim 10 wherein the disease is associated withdysregulation of angiogenesis.
 15. The method of claim 10 wherein thedisease is immune related.
 16. An isolated antibody that specificallybinds human hepatocyte growth factor activator, wherein the antibodycomprises a heavy chain CDR sequence comprising the respective H1, H2and/or H3 sequences depicted in FIG. 1A.
 17. The antibody of claim 16,wherein the H1, H2 and/or H3 sequences are as depicted in FIGS. 1B, 1Cand/or 1D.